AKR1B1 and AKR1B10, members of the aldo-keto reductase family of enzymes that participate in the polyol pathway of aldehyde metabolism, are aberrantly expressed in colon cancer. We previously showed that high expression of AKR1B1 (AKR1B1(HIGH)) was associated with enhanced motility, inflammation and poor clinical outcome in colon cancer patients. Using publicly available datasets and ex vivo gene expression analysis (n = 51, Ankara cohort), we have validated our previous in silico finding that AKR1B1(HIGH) was associated with worse overall survival (OS) compared with patients with low expression of AKR1B1 (AKR1B1(LOW)) samples. A combined signature of AKR1B1(HIGH) and AKR1B10(LOW) was significantly associated with worse recurrence-free survival (RFS) in microsatellite stable (MSS) patients and in patients with distal colon tumors as well as a higher mesenchymal signature when compared with AKR1B110(LOW)/AKR1B10(HIGH) tumors. When the patients were stratified according to consensus molecular subtypes (CMS), AKR1B1(HIGH)/AKR1B10(LOW) samples were primarily classified as CMS4 with predominantly mesenchymal characteristics while AKR1B1(LOW)/AKR1B10(HIGH) samples were primarily classified as CMS3 which is associated with metabolic deregulation. Reverse Phase Protein Array carried out using protein samples from the Ankara cohort indicated that AKR1B1(HIGH)/AKR1B10(LOW) tumors showed aberrant activation of metabolic pathways. Western blot analysis of AKR1B1(HIGH)/AKR1B10(LOW) colon cancer cell lines also suggested aberrant activation of nutrient-sensing pathways. Collectively, our data suggest that the AKR1B1(HIGH)/AKR1B10(LOW) signature may be predictive of poor prognosis, aberrant activation of metabolic pathways, and can be considered as a novel biomarker for colon cancer prognostication.